The present application claims the benefit of Argentina Application 000102102 filed May 3, 2000 and is incorporated herein by reference thereto.
The invention is related to continuous pharmacotechnical methods for the manufacturing of microcapsules of biodegradable and biocompatible polymeric materials that incorporate an active peptide in the polymer matrix by the formation of complex emulsions of the water/oil/water type (W/O/W). The process of the invention was develop to obtain these microcapsules in sterile injectable form thus allowing for the controlled administration, for adjustable release periods of between 1 to 18 weeks, of water soluble or dispersible drugs that are used for the treatment of neoplastic, gynaecological and other diseases. Thus, the invention is in the field of pharmacology and, particularly, it relates to pharmacotechnical processes for the manufacturing of injectable, controlled release medicines.
Since the pioneer work on encapsulation by coacervation conducted by B. K. Green (U.S. Pat. No. 2,800,457) directed to the development of copy papers, a number of publications and books have been written on microencapsulation of natural or synthetic substances into polymeric walls and their application in the controlled release of those substances (Microcapsule Processing and Technology, Asaji Kondo, 1979, Marcel Dekker). The gradual release of substances in controlled time intervals is important in pharmaceuticals drugs, foods, agrochemicals, fertilizers, and other products. A notable development, according to the number of publications observed in the recent years, took place in the area of microencapsulation of active pharmaceutical ingredients (Microspheres and Drug Therapy, Ed. Stanley S. Davis and others, 1984, Elsevier; Controlled Release Systems: Fabrication Technology, Vol I and II, Ed. Dean Hsieh, 1988, CRC Press, Inc.; Polymeric Drugs and Drugs Delivery Systems, Ed. Richard L. Dunn, 1991, ACS Symposium Series 469; Microencapsulation of Drugs, T. L. Whateley, 1992, Harwood; and Sustained Release Injectable Products, Ed. J. Senior and M. Radomsky, Interpharm Press, Denver, Colorado, USA, 2000). This complex physicochemical process has become a specialty field on its own rights.
In the area of pharmaceutical substances, clinical studies have shown that in many cases better therapeutic or pharmacological effects can be obtained by continuous infusion of the drug than when the same drug is administered by conventional methods, either in injectable, oral or other forms. Thus, it is necessary to consider using technologies for the prolonged release of active ingredients, which also include injection, oral, and other forms to administered the drug such as subcutaneous implants.
Generally, the substitution of a slow drug release method for a conventional one produces less pronounced collateral effects. These effects correlate to drug concentration peaks in the organism that occur when the minimum required active agent concentration is exceeded. One of those prolonged release methods is the use of microcapsules of polymers containing active agents such as polypeptides, proteins, hormones, nucleotides, and chemotherapy drugs, among others. Once the microcapsules are administered to the organism, drugs may be released by diffusion through a semi permeable wall in some cases, by wall dissolution in others, or by multiple mechanisms that include mainly the biodegradation of the encapsulating polymer in the living tissues into biocompatible fractions that follow a metabolic route for absorption or elimination. These polymer biodegradation processes cause, therefore, the slow dosing of the active ingredient.
Microcapsules based on re-absorbable and/or biodegradable polymers or co-polymers, have been the subject of extended research on manufacturing materials and methods, as well as on administration routes. Currently, microcapsules are increasingly applied in the administration of biotechnology products, including water soluble, slightly soluble, or insoluble substances. There are several administration routes for this particular type of microcapsules, depending on the drug to be released. The microcapsules can be adapted to injectable administration as well as to the administration to the gastrointestinal system, nasal tissues and other access routes.
Provided they degrade into biocompatible residues, a large number of polymers with a main hydrophobic chain may be used to form the microcapsule wall. Occasionally, the polymers may require a special level of purification. Among others, generally used biodegradable polymers are: poly(d,l-lactic) acid; poly(d,l-lactic-glycolic) copolymer; poly(caprolactones); poly(hydroxybutirate); poly(orthoesters); and poly(anhydrous) as well as mixtures of these and other polymers (Polymeric Drugs and Drug Delivery Systems, Ed. Richard L. Dunn, 1991, ACS Symposium Series 469, p. 15-20).
Poly(d,l-lactic-glycolic) acid, a d,l-lactic acid and glycolic acid copolymer, generally known as PLGA, and the homopolymer of d,l-lactic acid, poly(d,l-lactic) acid, generally known as PLA, have been used since 1973 as polymers for medicine microcapsules. Among others, examples of its use are: the microencapsulation of a narcotic antagonist like naltrexone (J. H. R. Woodlnad et al., J. Med. Chem., vol 16, 897, (1973); S. E. Harrigan et al., Midl. Macromol. Monogr., vol 5 (Polym. Delivery Systems), vol 91 (1978)); of anaesthetic substances (N. Wakiyama et al., Chem. Pharm. Bull., vol 30, 3719, (1982)), and of steroids (D. L. Wise et al., J. Pharm. Pharmacol., vol 32, 399, (1980)). We can specially mention the use of PLGA 50:50 and 69:31 (mole ratio of lactic acid to glycolic acid) in the microencapsulation of nafarelin acetate, an analog of the luteinizing hormone release hormone (LH-RH) (L. M. Sanders et al., J. Pharm. Sci., vol 73, 1294-1297, (1984)). Currently, it is completely accepted the use of PLGA and PLA as biocompatible polymers that are degradable to toxically acceptable products that are eventually eliminated from the body (D. H. Lewis, Biodegradable Polymers as Drug Delivery Systems, Ed. M. Chasin et al., Marcel Dekker, New York, NY, pp 1-42, 1990).
PLA or PLGA of controlled molecular weight are obtained by polycondensation of cyclic dimers of the lactic and glycolic acids, known as lactide and glycolide. There is an extensive literature on synthesis and purification methods of PLA and PLGA with molecular weights of 25000 Daltons or less. Among direct polycondensation procedures it can be mentioned those that are carried out without a catalyst, those that use a metallic catalyst as described in, among others, U.S. Pat. Nos. 3,297,033, 3,773,919 and 3,839,297, and those that use acid catalysts such as ionic exchange resins as taught in U.S. Pat. No. 4,273,920.
Slow release microcapsules are known in the administration of hormones, antibiotics, anti-inflammatory substances, antitumoral drugs, antihypertensive drugs, antipyretics, vasodilators, antiallergic agents, and analgesics, where PLGA o PLA is the constitutive biodegradable wall material.
Of particular interest for the purpose of the present invention are microcapsules containing biologically active substances that are either water soluble or can form a suspension in an aqueous phase. Among the water soluble drugs of interest are active peptides and specially hormones. One water soluble hormone of particular interest is leuprolide acetate, which was synthesized almost simultaneously by J. A. Vilchez-Martinez et al., (Biochem. Biophys. Res. Commun. 59, 1226, (1974)) and by Fujino et al. (M. Fujino et al., Biochem. Biophys. Res. Commun. 60, 406-413, (1974)), and it is the first superactive agonist of the luteinizing hormone release hormone (LH-RH), with approximately 10 times the biological activity of LH-RH. It has been used for the treatment of hormone dependent tumors in prostate (T. W. Redding et al., Proc. Nat. Acad. Sci. USA, vol 78, 6509-6512, (1981)) and breast cancers (E. S. Johnson et al., Science, vol. 194, 329-330, (1976)), endometriosis (D. R. Meldrum et al. J. Clin. Endocrinol. Metab., vol. 54, 1081-1083, (1982)) and uterine fibrosis (M. Filicori et al., Am. J. Obstet. Gynecol., vol. 152, 726-727, (1985)).
In studies conducted by H. Okada et al. on vaginal absorption of leuprolide in rats, it was observed that constant levels of the drug in the blood produce higher castration rates than the intermittent and pulsating administration of this drug. It was then thought that a slow release injection should produce optimal therapeutic results (H. Okada et al., J. Pharm. Dyn., vol. 6, 512-522, (1983)). This thought originated the development of the so-called depot injection method, that allows for a leuprolide acetate release period of up to 120 days(H. Okada et al. Jap. Patent Appl. No. 207760, U.S. Pat. No. 4,652,441; Y. Ogawa et al. Chem. Pharm. Bull., vol 36, 1095, (1988)). Other hormones of particular interest for the present invention, agonists of the luteinizing hormone releasing hormone (LH-RH) are: goserelin acetate (U.S. Pat. No. 4,100,274), buserelin acetate (U.S. Pat. No. 4,024,248), triptorelin acetate (U.S. Pat. No. 4,010,125) and nafarelin acetate (U.S. Pat. No. 4,234,571)
A number of methods have been developed for the microencapsulation of active ingredients into biodegradable and non-biodegradable polymers. Among these methods, three main types predominate: those based on emulsion/separation of phases; those based on xe2x80x9csprayxe2x80x9d drying; and those based on evaporation of the solvent of an aqueous or organic vehicle phase.
In emulsion/separation of phase techniques, an aqueous solution of the drug, or the drug in powder state, is dispersed into an organic solution containing the polymer. Once the emulsion is formed, a coacervation agent is added, generally a vegetal or mineral oil, which induces the formation containing the active ingredient. See, e.g., U.S. Pat. Nos. 4,675,189 and 4,835,139. These methods have the disadvantage of using large amounts of solvents and oils. In addition, the microcapsule formation stage also depends on the quantities of polymer, solvent, and coacervation agent used. An additional undesirable effect is the tendency of the particles to adhere to each other during the manufacturing process.
Encapsulation by xe2x80x9csprayxe2x80x9d drying consists in initially preparing an aqueous phase containing the active agent in solution or suspension. This aqueous medium is dispersed into an organic phase that contains the polymer to produce a water/oil (W/O) type emulsion. This emulsion is pulverized in a hot air flow in a drying equipment. The microcapsules are formed by the evaporation of the organic solvent. U.S. Pat. No. 5,622,657 teaches one application of this method for the semi-continuous manufacturing of peptide microcapsules, including leuprolide acetate. The patent teaches the formation of microspheres by xe2x80x9csprayxe2x80x9d drying of a water/oil emulsion and, simultaneously, spraying from an auxiliary nozzle an aqueous solution containing a substance that contributes to prevent particle adherence during their formation.
Procedures based on solvent evaporation of an aqueous or organic phase are the most common one in microcapsule manufacture. The basic technique consists in dispersing the drug in a polymer solution in an organic solvent. The active ingredient may be in powder form or dissolved in a solvent that is emulsionable in the polymer solution. This first dispersion is then emulsioned in a second solvent, which is called the vehicle solvent, that is non miscible with the solvent in the polymer solution. This last solvent is then evaporated in a subsequent step of the process.
There are a variety of techniques based on solvent evaporation that were developed for the microencapsulation of water soluble and non soluble substances.
U.S. Pat. No. 3,691,090 teaches the encapsulation of water soluble substances, including medicines, where these substances are dispersed into an organic solvent which is either miscible or partially miscible in water. The polymer is dissolved in that solvent and the organic phase is emulsified into an aqueous medium containing an inorganic salt to prevent solubilization of the organic solvent. The resulting oil/water (O/W) emulsion contains oily microspheres of polymer containing the active substance. Microcapsules are consolidated by organic solvent evaporation.
U.S. Pat. No. 3,960,757 teaches a method of encapsulation of water insoluble or slightly soluble medicines consisting in dissolving or dispersing the active substance into a polymer solution in an organic solvent which is almost insoluble in water. The organic solvent must have a vapor pressure greater than water. The organic phase is emulsified in a vehicle consisting of an aqueous solution of a hydrophilic colloid or a surfactant agent, to produce an oil/water (O/W) two-phase system. The organic solvent is then removed by evaporation and the microcapsules consolidate. The patent also teaches the use of gelatin, polyvinyl alcohol (PVA), carboximethylcellulose, and other substances, as hydrophilic colloids. The patent teaches to use as organic solvent to dissolve the polymer certain chloroalkanes such as dichloromethane, ethylene chloride, chloroform, and others. The polymers used in the taught process are of the hydrophilic type.
U.S. Pat. No. 5,540,973 teaches a process to prepare microspheres containing the LH-RH hormone and its analogs in a biodegradable and water insoluble polymer matrix. According to the taught process, the polymer is dissolved in a first organic solvent, and then the hormone is dispersed in that solution by agitation. Then, this first solvent is evaporated to dryness, and the residual mass is contacted with a second solvent where the polymer dissolve, but not the active drug which stays in suspension. The final stage comprises the preparation of an oil/water (O/W) emulsion, the addition of a surfactant agent, and the evaporation of the second solvent to cause the formation of microspheres.
Of particular interest for the present invention is the procedure of microencapsulation that uses a in-liquid drying process, or complex emulsion method, as the method was called by Asaji Kondo (Microcapsule Processing Technology, 1979, Marcel Dekker, Ch. 10, p. 106), and more specifically, the in-water drying method. See Japanese Patents Nos. JP39-28744, JP42-13703, and JP43-10863 and French Patent No. FR1362933. This method consists in first preparing an aqueous phase in oil emulsion (W/O) and then forming for encapsulation a second emulsion ([(W/O)/W] type emulsion) by dispersing the first water in oil emulsion in a second aqueous phase.
This method has a number of advantages: it doesn""t need pH adjustments, the use of a significant heat source, or any special reactant. Thus, chemically unstable materials can be microencapsulated without substantial degradation. Other advantages, that are dependent on the degree of control that mat be had on the physicochemical conditions of the preparation, are: better yields of microcapsules free from agglomeration, and better efficiency in active ingredient encapsulation, compared to the other methods described above. Further, this process can be used to prepare small batches (0.25 to 1.0 g) of active ingredient batches, useful when the active ingredient is very expensive. In addition, the process can be easily scaled-up to process larger amounts (10 to 100 g) of active ingredient.
Essentially, microencapsulation by in-water drying of complex emulsions consists in preparing a first water in oil type emulsion (W/O) by dispersing a volume V of an aqueous solution of the active material into an eight times V volume of a solvent partially or totally immiscible in water, where the polymer that will form the microcapsule wall was dissolved. This solvent must have a boiling point lower, and a vapor pressure greater than water, so it can be evaporated in presence of water. Separately, it is prepared a 40 times V volume of an aqueous solution containing a stabilizer or protective colloid. Microencapsulation is caused by agitating the last solution while adding the (W/O) dispersion, to obtain a total volume approximately equal to 50 times V of a water in oil in water double emulsion [(W/O)/W]. This system is stable. The fluid microcapsules are made of an organic solution of the polymer containing dispersed in its interior micro- and nano-drops of an aqueous solution of the active ingredient. This polymer organic solution is emulsified in the external aqueous phase. When the organic solution polymer is dried by heating and/or reduced pressure, the polymeric matrix that forms the microcapsule becomes hard, and the aqueous micro-drops or nano-drops of the active ingredient remain trapped into the microcapsule.
The microcapsule size and stability are influenced mainly by factors such as (W/O) emulsion viscosity, local agitation intensity, temperature, and the addition of some additive substances in the aqueous phases. Using this method microcapsules of 1 to several hundred of microns in diameter may be prepared. It is convenient, in some applications, to add to the first (W/O) emulsion certain hydrophilic substances such as, among others, albumin and gelatin, dissolved in water to act as retention agent of the active substances. (French Patent No. FR1362933; Japanese Patent No. JP43-10863). These hydrophilic substances contribute to stabilizing the (W/O) emulsion by preventing micro-drop coalescence. Further, it is advisable, during preparation of the second [(W/O)/W] emulsion, to dissolve in the external aqueous phase a hydrophilic protective colloid such as gelatin or polyvinyl alcohol (PVA) to function as stabilizer. (French Patent No. FR1362933; Japanese Patent No. JP42-13703; A. Kondo, Ind. Chem. (Japan), 72 (2), 493 (1969)). These colloids must be only slightly soluble in the organic solvent of the oily phase where the first (W/O) emulsion is produced (W/O). If no protective colloid is used, the active agent entrapment in the microcapsules is notably reduced, and a microcapsule inversion may occur. This is a particular situation where the aqueous internal core is released to the external aqueous medium, and only empty polymer microspheres are formed. The process results depend strongly on the selection and specific molecular properties of the hydrophilic protective colloid used in the second emulsion as well as the active ingredient retention substance used in the first emulsion. However, patents and scientific publications, although mentioning a number of possible substances that can be used for these purposes, do not give any significant specifications about these substances.
A disadvantage of in-water drying is that it takes a long time to eliminate the solvent from the polymer solution, which includes the micro-drops containing the active ingredient. If the solvent is removed too rapidly, little orifices and bubbles may be formed on the surface of the microcapsule walls. One way to ameliorate these problems is to extract the organic solvent with another solvent which is soluble in water and the organic solvent but does not dissolve the polymer (Gevaert, Photo-Production N. V., French Patent No. FR1362934). Another way to reduce the problems is to conduct a controlled evaporation of the solvent by gradual heating combined with pressure reduction.
In complex emulsion in-water drying, it is preferable that the organic solvent and the polymer not be miscible with the active ingredient, so it could be encapsulated. The active ingredient may be in aqueous solution or dispersion, or as solid powder. In an aqueous solution, if the dissolved active drug has a low molecular weight, it will tend to diffuse through the microcapsule wall during the encapsulation process. On the other hand, if it is a molecular substance with a molecular weight of several thousand Daltons, it will be retained inside the microcapsule.
The complex emulsion in-water drying method for encapsulation of highly hydrophilic pharmaceutical drugs is frequently used. Discontinuous procedures to obtain prolonged release microcapsules for injectable use, for implants, and transdermal or oral administration are described in, among others, European Patent No. EP0765659, and U.S. Pat. Nos. 4,652,441, 4,954,298, 5,271,945, 5,330,767, 5,611,971, and 5,651,990.
Discontinuous procedures for encapsulation of water soluble peptides for pharmaceutical uses, using this complex emulsion in-water drying method and PLGA or PLA as encapsulating polymers, show some drawbacks such as high dispersion of particles sizes that range from 1 to more than 400 microns, micro particles adhesion, process control difficulties, and poor reproducibility.
To prepare the first emulsion (W/O) in a discontinuous process requires variable intensity of agitation and mixing time that are dependent not only on the size of process, but also on other variables such as size and shape of the mixer. Because of the high viscosity of the phases, a good agitation or mixing of the total mass cannot be obtained. The shear forces applied by the mixing element (agitation turbines, dispersers, or ultrasound) can be transmitted only a few millimeters from the applying point. The result is a high dispersion of particle size in the first emulsion (W/O).
The preparation of the second emulsion, where the total external aqueous phase is placed in one reactor and the first emulsion (W/O) is added slowly to form the complex emulsion [(W/O)/W], is strongly dependent on factors such as: time in adding the phases, temperature, initial volume of first emulsion to second emulsion ratio, polymer concentration in the organic phase, nature and concentration of the protective colloid in the second aqueous phase, and position of the injection point of the (W/O) emulsion. Consequently, the control of this discontinuous process is extremely complicated. The results are high dispersion of particle size and low yields of microencapsulated material which passes through a mesh 200 (75 microns), the maximum size suitable for injectable preparations. It has been observed that traditional discontinuous manufacturing processes yield near to 30% of microcapsules having a diameter greater than 75 microns.
When the second emulsion is formed by adding the first emulsion over a total volume of the aqueous phase where the microcapsules will be formed, an important factor is the location of the first emulsion injection point. When the external aqueous phase is strongly agitated, particles of different sizes may be formed practically in the entire volume of the external aqueous phase. When these particles reach a size such that the surface evaporation of the volatile solvent allows the hardening of the microcapsule, this microcapsule can no longer reduce its size whatever be the length of the agitation time. The result is a wide dispersion in final particle size: microcapsules formed farther away from the point of application of shear forces are of a larger size whereas those formed in the neighborhood of the shear force application point are of a smaller size.
As it was mentioned previously, microcapsules consolidate when the superface evaporation of the volatile solvent used to dissolve the polymer hardens the surface to such an extent that is no longer possible the subdivision of microcapsules into smaller size particles. This evaporation is strongly dependent on the water ability to eliminate the organic solvent by absorption. For example, methylene chloride reaches a solubility of about 1.3% by weight at room temperature. In a discontinuous system this absorption capacity is time-dependent. At the beginning of the operation the microcapsules are formed in a medium where there is only water with a protective colloid with tensoactive activity. On the other hand, at the end of the mixing, the microcapsules are in a complex water-tensoactive agent system with increasing amounts of solvent and microcapsules. In this last situation, there is an increased probability of particle agglomeration.
Several of the procedures described in the literature include, after the separation of the external aqueous phase, the steps of washing the microcapsules with water followed by drying to remove moisture, milling and sieving of the dried product to eliminate particle agglomerates and homogenize its granulometry and, finally, the dosing of the solid to obtain the final product. These operations with solids present the same difficulties and demand the same care that are typical of operations with injectable pharmaceutical powders. Expensive equipment must be used to ensure sterile conditions, and prevent contamination and moisturizing of the microcapsules since the material is extremely hydrophilic, presents a high specific surface and must contain no more than 1% of water.
Further, in discontinuous methods, active peptide losses can reach up to 70% for the full process. These losses are calculated by comparing the amount of active peptide used as raw material and the amount retained in those microcapsules of suitable size to be used as an injectable product. The losses include the amount of active peptide that is entrapped in microcapsules larger than 75 microns, plus the amount that is dissolved in the non-emulsified medium, plus the amount lost during the washing step, plus the amount entrapped in very small microcapsules that also go away during washing.
This invention relates to a novel process for producing microcapsules for the sustained release, in adjustable release periods, of water soluble peptides. The process comprises the following steps: (1) continuously intermixing an aqueous solution of a water-soluble active peptide and a retention substance with an oily solution of a biodegradable polymer in an organic solvent that is insoluble or only slightly soluble in water, in a first mixer closed to the atmosphere to produce a first emulsion; (2) cooling the emulsion; (3) continuously intermixing the emulsion and an aqueous phase containing a protective hydrophilic colloid in a second mixer also closed to the atmosphere to produce a second emulsion; (4) removing the organic solvent from the second emulsion in a closed vessel to produce microcapsules containing the water soluble peptide; (5) adjusting the size distribution of the microcapsules; (6) dispersing the microcapsules in an aqueous medium containing a lyophilization excipient; (7) distributing the aqueous dispersion of microcapsules into vessels and freezing the medium at a temperature of less than about 20xc2x0 C.; and (8) lyophilizing the frozen microcapsule dispersion.
The invention also relates to microcapsules produced according to the above described novel process. The invention also relates to the use of these microcapsules in the manufacture of formulations for the sustained release of materials.
The process of this invention reduces the number of operation steps of the process; improve the reproducibility of the process variables and, thus, facilitates the control of the process; produces a narrow and reproducible particle size distribution, composition and internal distribution of the active agent and, thus, facilitates the production of microcapsules with controlled release periods of the active drug; ensures a high retention of the active material inside the, microcapsules and, thus, minimizes losses of expensive raw materials; substantially improves the yield of particles with the desired size; minimize product exposure to the atmosphere during the process steps and, thus, decreases equipment requirements (and associated costs) to ensure sterile condition and low contamination; and improves the quality of the lyophilized product. Thus, the novel process substantially improves manufacturing productivity in comparison to existing processes and results in a product of higher quality.